von Willebrand disease (vWd) subtypes with different structural abnormalities of von Willebrand factor (vWf) have been identified and a vWf tryptic peptide has been isolated which contains the platelet GPIb binding domain. The subunit structure of normal and vWd plasma vWf was examined by SDS polyacrylamide gel electrophoresis and monoclonal antibody immunoblotting following rapid purification on an analytical scale in the presence of protease inhibitors. Of the vWd subtypes with abnormalities of vWf multimeric structure, two categories were identified: those with increased proteolytic cleavage of vWf, as compared to normal, and those in which cleavage was minimal or absent. The hypothesis that these differences are the result of altered susceptibilities of the variant molecules to proteolysis will be tested. For vWf subtypes in which loss of large multimers proves to result from proteolysis, the specific cleavages responsible will be identified. Peptides composed of amino acids which bridge cleavage sites will be synthesized and antibodies will be prepared against them. These antibodies and/or the peptides themselves will be assessed for their ability to block cleavage by enzymes shown to cause loss of large multimers. In addition, the molecular basis for both categories of variant vWf molecules will be explored by two dimensional peptide analysis and immunoblotting with monoclonal antibodies known to react with peptides important to vWf function. One such peptide is the 52/48 kDa tryptic fragment we have isolated at the initial step in defining the structural requirements for the platelet GPIb binding domain of vWf. This doublet migrates as a single 46 kDa species after 70% deglycosylation. After reduction, alkylation and extensive denaturation it retains the ability to block binding of intact vWf to GPIb. Two anti-vWf monoclonal antibodies which are potent inhibitors of vWf binding to GPIb react with a 13 kDa cyanogen bromide fragment of this peptide on immunoblotting. Based on the amino acid sequence of the 13 kDa species, overlapping peptides will be synthesized to identify the sequence critical to GPIb binding. A similar approach will be used to characterize the vWf binding domains for Factor VIII procoagulant protein, collagen, and in the GPIIb/IIIa platelet receptor.